About 70 - 80 % of hepatitis C virus (HCV) infections result in chronic infection that can lead to severe liver diseases, including cirrhosis and hepatocellular carcinoma (HCC). Studies have shown that HCV evades the type I interferon (IFN) response of the host by interfering with pathogen-recognition receptor (PRR) signaling. HCV NS3/4A protease inhibits RIG-I and toll-like receptor 3 signaling by proteolytic inactivation of IPS/MAVS/VISA/CARDIF and TRIF, respectively. Nevertheless, the mechanism by which HCV evades the host innate immunity to establish persistent infection and induces pathogenesis has not been completely characterized, and recently, an NS3/4A-independent suppression of PRR signaling has been described although the mechanism remains unknown. Recent data from this laboratory, using the JFH1 HCV sequence of genotype 2a that produces infectious virus particles in Huh7 human hepatoma cells, indicate the presence of another viral element that suppresses PRR signaling, independent of NS3/4A. The viral element is the previously described F/ARF protein, generated by -2/+1 ribosomal frameshift at the A-rich site at codons 8 - 14 of the HCV core-coding sequence. Mutations that abolished -2/+1 frameshift enabled IFN2 and IFN1 induction by HCV RNA, and a synthetic double strand RNA, poly(IC);and F/ARF protein expression could suppress IFN2 induction by poly(IC). Furthermore, the modulation of the type I IFN response required retinoic-acid-inducible gene I (RIG-I), suggesting that the intracellular PRR, RIG-I/MDA-5, was/were being affected. JFH1 supported -2/+1 frameshift at codons 8 - 14, and a monoclonal JFH1 F/ARF antibodies- reactive protein was detected in JFH1 F/ARF-transfected cells, JFH1-replicating cells, and HCV-infected patient liver that was absent in the controls. Therefore, F/ARF protein may participate in the suppression of RIG-I/MDA-5 signaling during persistent HCV infections. The goal of this study, therefore, is to determine the role of F/ARF and other -2/+1 protein products in the modulation of PRR signaling by HCV. The specific aims are: 1) to identify and characterize the viral element(s) derived from the -2/+1 frame of HCV core-coding gene that modulate(s) RIG-I/MDA-5 signaling pathway, independent of NS3/4A, using JFH1 sequence of genotype 2a. We will continue to characterize the suppression of PRR signaling and viral elements that are involved, using standard molecular biology approaches as well as mass spectrometry. 2) To determine the mechanism of inhibition of RIG-I/MDA-5 signaling by these element(s). The signaling step(s) that is/are suppressed by the element(s) will be identified as well as the mechanism(s). At the end of the study, novel modulator(s) of type I interferon response and biological function(s) of HCV alternate frames decoding would be identified. The long-term goal is to determine the mechanism by which HCV evades host immune response to improve the efficacy of the existing therapy or to develop new anti-HCV agents.